CN101285060B - Process of chitosan-arginine resin anion immobilizing chymotrypsin - Google Patents
Process of chitosan-arginine resin anion immobilizing chymotrypsin Download PDFInfo
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Abstract
The invention discloses a method for immobilizing chymotrypsin by chitosan-arginine anionic resin, relating to a method for immobilizing enzyme by a chitosan derivative. The method is to make cross-linked chitosan resin with commercial chitosan as raw material first, then to make chitosan-arginine anionic resin, and finally to perform Schiff reaction to immobilize chymotrypsin to obtain finished products. The method has the advantages of simpleness, easy operation, moderate reaction condition, low production costs, high stability of immobilized chymotrypsin, high recovery of enzyme activity up to 89.95 percent, long half value period which is 18 hours at 75 DEG C and 56 days at 4 DEG C, with optimal pH of 5.92. Products made by the method can be widely used in enzymic reaction in aqueous or nonaqueous medium in medicine, food, light manufacturing and other industries.
Description
Technical field
The invention belongs to the enzyme immobilization technology field, particularly the method for chitosan derivatives immobilized enzyme.
Technical background
Studies show that: Quimotrase pharmaceutically has digestion pus and necrotic tissue, the clean surface of a wound that disappears, and local anti-inflammatory promotes absorption of hematoma, effects such as wound healing; Have functions such as decomposing protein, leather depilation in industries such as food, light industrys.At present, the resolvase of use mainly contains the cost height, and utilization ratio is low, and the useless enzyme of discharging is shortcoming such as contaminate environment also.Therefore, long novel immobilizing chymotrypsin of transformation period is used in development, has important economy, social value.
Chitosan is abundant, the cheap and easy to get natural macromolecular material of nature content, because of it has good film forming, preserves moisture, absorption, characteristic such as antibiotic, is widely used in the industries such as medicine, food, light industry.Again because of chitosan has readily biodegradable, safety non-toxic, good biocompatibility carries out characteristics such as chemically modified, particularly chitosan derivatives possess hydrophilic property are good, rigidity is big, mechanicalness is good easily, is the carrier of excellent immobilized enzyme.
The method of existing immobilized enzyme comprises entrapping method, absorption method, covalent attachment method and crosslinking etc.Entrapping method is meant enzyme is embedded in trickle grid of high-molecular gel or the semi-permeable membranes, its preparation technology is easy and reaction conditions is comparatively gentle, can obtain higher enzyme activity reclaims, owing to have only small molecules can pass through high-molecular gel network, and diffusional resistance can cause enzyme activity to reduce, so entrapping method only is fit to act on the enzyme of small molecules substrate and product.The absorption rule is meant that enzyme is adsorbed on the process for fixation of insoluble carrier, and this method is easy and simple to handle, mild condition, and the vigor of enzyme loses seldom, and carrier is cheap and easy to get, can use repeatedly, still, a little less than enzyme and the carrier interactions power, easily comes off.The covalent attachment method is meant enzyme and the carrier process for fixation with covalent bonds, the immobilized enzyme of this method, and difficult drop-off can use considerable time continuously, but the activation more complicated of carrier, and combined techniques has fierce reaction and make the enzyme activity loss bigger.Crosslinking refers to make process for fixation crosslinked between enzyme and the carrier with difunctional or poly functional reagent, crosslinking immobilized enzyme good stability, " chitosan-immobilized tryptic preparation and physico-chemical property research " literary composition as University Of Ningbo's journal the 1st phase of March in 1999, the chitosan that obtains from the chitin crude product is disclosed, again in the method that under the effect of linking agent glutaraldehyde trypsinase is fixed on this chitosan; The weak point of this method is to be difficult to obtain purified chitosan from the chitin crude product, and directly with chitosan as carrier, the trypsinase macromole near the time steric hindrance big, direct crosslinked immobilized enzyme efficient is low, causes trypsinase molecule self-crosslinking easily.And for example " a kind of preparation method of nano-magnetic chitin immobilized enzyme " patent of publication number CN1904043A discloses and has used the surface to contain the method for nano-magnetic chitin and zymoprotein molecule prepared in reaction immobilized enzyme under the action of a magnetic field of acid chloride groups; The nano-magnetic chitin carrier that the weak point of this method is to prepare certain particle diameter is difficulty relatively, and nano-magnetic chitin is in use oxidized easily and take place heavy poly-ly simultaneously, causes the reduction of enzyme activity.
Summary of the invention
The objective of the invention is weak point, a kind of method of chitosan-arginine resin anion immobilizing Quimotrase is provided at existing chitosan resin immobilized enzyme method.Characteristics such as that this method has is simple to operate, enzyme is lived rate of recovery height, reaction conditions gentleness, preparation cost are low, the free Quimotrase enzyme heat stability of immobilizing chymotrypsin increases simultaneously, the applicable pH value reduces, transformation period prolongs, and can recycle, reduce and pollute, make full use of resource.
Mechanism of the present invention: owing to contain a large amount of free amine groups in the chitosan molecule chain, these amino have lone-pair electron, has very strong nucleophilicity, and glutaraldehyde is a kind of bifunctional reagent commonly used, can generate schiff base structure with the amino generation crosslinking reaction of chitosan, crosslinked mainly is in intermolecular generation; Dicyclohexylcarbodiimide (DCC) is a kind of peptide condensing agent that connects commonly used, and it can activated carboxyl, promptly strengthens the Electron Affinities of carboxyl carbon atom, makes nucleophilic nonionic amino be easy to it is attacked, and generates peptide bond thereby condensation reaction takes place.Once more with glutaraldehyde as linking agent, the remaining amino shiff of generation of Quimotrase surface free amine group and arginine is reacted, promptly get the chitosan-arginine resin anion immobilizing Quimotrase.
The object of the present invention is achieved like this: a kind of method of chitosan-arginine resin anion immobilizing Quimotrase, with commercially available chitosan is raw material, prepare crosslinked chitosan microsphere earlier, the refabrication chitosan-arginine resin anion, carry out the schiff's reaction immobilizing chymotrypsin at last and finished product.Concrete method steps is as follows:
(1) preparation crosslinked chitosan resin
With commercially available chitosan is raw material, and earlier chitosan being added the hydrochloric acid mass percent is in 1～5% dilute hydrochloric acid, after the stirring and dissolving, just prepares chitosan mass percentage ratio and be 1～4% chitosan acidic sol.Back in the chitosan acidic sol: the volume ratio of whiteruss is 1: 1～4 ratio, slowly add the chitosan acidic sol in the whiteruss, stir 20～30min, and after heat temperature raising to 35～45 ℃ make chitosan paraffin solution, add again that to account for chitosan paraffin liquor capacity mark be 0.05～0.25% tween 80, carry out emulsification 20～30min, add then that to account for chitosan paraffin liquor capacity mark be 0.05～0.3% glutaraldehyde cross-linking agent, make the amino participation of the part crosslinking reaction of chitosan molecule, thereby make linear chitosan molecule be transformed into chitosan microball.Add again that to account for chitosan paraffin liquid quality fraction be 0.01～0.1% lime carbonate pore-creating agent, carry out the suspension that drilling reaction 50～70min makes chitosan microball.PH value with sodium hydroxide solution adjusting chitosan microball suspension is 9～10 at last, and is heated to 55～75 ℃, stirs and solidifies 2～4h, refilters, and abandons filtrate, collects filter residue.Earlier with washing with alcohol 3～6 times, the back is a lime carbonate in 1～5% diluted hydrochloric acid dissolution filter residue with massfraction, is washed with distilled water to neutrality again, just prepares crosslinked chitosan resin to filter residue.
(2) preparation of chitosan-arginine resin anion
(1) step finish after, go on foot the crosslinked chitosan resin of preparing with (1), adding the arginine massfraction is in 0.5～2.0% arginine solution, vibration 20～30min, chitosan resin is suspended in the arginine solution, prepares the chitosan mass mark and be 0.5～2.5% chitosan-arginine solution.Then in the arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is 1: 0.5～2.5 ratio, in chitosan-arginine solution, add DCC and connect the peptide condensing agent, carry out condensation reaction 20～40min, make the amino and arginic carboxyl generation condensation reaction of another part in the chitosan resin, just prepare the chitosan-arginine resin.At last after filtration, abandon filtrate, collect filter residue.Filter residue is used washing with alcohol 3～6 times earlier, and the back refilters after using distill water dialysis 8～12h, abandons filtrate again, regathers filter residue, just prepares chitosan-arginine the moon from resin.
(3) Quimotrase is fixing
After (2) step finished, earlier Quimotrase being added the pH value was in 4.5～6.0 phosphate buffered saline buffers, stir, and with regard to the volumetric molar concentration of preparing Quimotrase the Quimotrase phosphate solution of 0.05～0.08mol/L.The chitosan-arginine resin anion that prepared in (2) step the back: the mass ratio of Quimotrase is 1: 15～25 ratio in the Quimotrase phosphate solution, chitosan-arginine resin anion is added in the Quimotrase phosphate solution, under 30～60 ℃ water bath with thermostatic control condition, vibration 20～40min is suspended in the Quimotrase phosphate solution chitosan-arginine resin anion.Again in glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 6～14 ratio, glutaraldehyde is joined in the Quimotrase phosphate solution of suspension chitosan-arginine resin anion, and place vibrator to vibrate, after carrying out schiff's reaction 50～70min, carry out suction filtration, abandon filtrate, collect the resin filter residue, the resin filter residue is washed 4～7 times with phosphate buffered saline buffer, remove free Quimotrase, suction filtration is abandoned filtrate more again, regather chitosan-arginine negatively charged ion immobilizing chymotrypsin filter residue and finished product.
After the present invention adopts technique scheme, mainly contain following characteristics:
(1) immobilization effect is good.Owing to have a long fat chain structure-(CH between guanidine radicals and the carboxyl in the arginine molecule
2)
4After chitosan resin and arginine reaction, be equivalent to connect many " cantilevers " to chitosan resin, with its carrier as immobilized enzyme, steric hindrance is big when having avoided the zymoprotein macromole near carrier, direct crosslinked immobilized enzyme efficient is low, and easily causes shortcomings such as enzyme molecule self-crosslinking, so the immobilized enzyme better effects if; Carry out enzymatic reaction with this immobilized enzyme, be more conducive to immobilized enzyme and contact, improve reaction efficiency with reaction medium.
(2) stability of immobilized enzyme is high.The inventive method is to adopt fixedly Quimotrase of glutaraldehyde cross-linking method, increase the bonding force between enzyme and the carrier, improve the physical strength of immobilized enzyme, avoid that the enzyme molecule makes the stability of enzyme obtain further enhancing from the defective that carrier comes off in the use.
(3) the applicable pH value reduces.The optimum pH of free Quimotrase is 8.0, and the optimum pH of immobilizing chymotrypsin only is 5.92, has further enlarged the use range of Quimotrase.
(4) production cost is low.The Quimotrase of the inventive method employing chitosan-arginine resin anion immobilizing is easier to be separated with organic solvent, more helps the recycling of enzyme, improves the service efficiency of enzyme, reduces production costs.
(5) enzyme rate of recovery height alive, long half time.Immobilized chymotrypsin protein enzyme heat stability increases, and enzyme is lived the rate of recovery up to 89.95%.Transformation period prolongs, and promptly 75 ℃ of transformation period reach 18 hours, and 4 ℃ of transformation period reach 56 days.
(6) the inventive method is simple, and is easy and simple to handle, and the reaction conditions gentleness is easy to utilize in the production process.
The chitosan-arginine resin anion immobilizing Quimotrase that adopts the inventive method to prepare, extensively method is applied to the various waters of industries such as medicine, food, light industry and the enzymatic reaction of nonaqueous phase.
Embodiment
Below in conjunction with embodiment, further specify the present invention.
Embodiment one
A kind of concrete steps of method of chitosan-arginine resin anion immobilizing Quimotrase are as follows:
(1) preparation crosslinked chitosan resin
With commercially available chitosan is raw material, and earlier chitosan being added the hydrochloric acid mass percent is in 2% dilute hydrochloric acid, after the stirring and dissolving, just prepares chitosan mass percentage ratio and be 2% chitosan acidic sol.Back in the chitosan acidic sol: the volume ratio of whiteruss is 1: 1 a ratio, slowly add the chitosan acidic sol in the whiteruss, stir 25min, and after heat temperature raising to 40 ℃ makes chitosan paraffin solution, add again that to account for chitosan paraffin liquor capacity mark be 0.1% tween 80, carry out emulsification 25min, add then that to account for chitosan paraffin liquor capacity mark be 0.15% glutaraldehyde cross-linking agent, make the amino participation of the part crosslinking reaction of chitosan molecule, thereby make linear chitosan molecule be transformed into chitosan microball.Add again that to account for chitosan paraffin liquid quality fraction be 0.05% lime carbonate pore-creating agent, carry out the suspension that drilling reaction 60min makes chitosan microball.PH value with sodium hydroxide solution adjusting chitosan microball suspension is 9.5 at last, and is heated to 60 ℃, stirs and solidifies 3h, refilters, and abandons filtrate, collects filter residue.Earlier with washing with alcohol 5 times, the back is a lime carbonate in the 3% diluted hydrochloric acid dissolution filter residue with massfraction, is washed with distilled water to neutrality again, just prepares crosslinked chitosan resin to filter residue.
(2) preparation of chitosan-arginine resin anion
(1) step finish after, with the crosslinked chitosan resin that (1) step prepared, adding the arginine massfraction is in 1% arginine solution, vibration 25min, chitosan resin is suspended in the arginine solution, prepares the chitosan mass mark and be 1.5% chitosan-arginine solution.Then in the arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is 1: 1 a ratio, in chitosan-arginine solution, add DCC and connect the peptide condensing agent, carry out condensation reaction 30min, make the amino and arginic carboxyl generation condensation reaction of another part in the chitosan resin, just prepare the chitosan-arginine resin.At last after filtration, abandon filtrate, collect filter residue.Filter residue is used washing with alcohol 4 times earlier, and the back refilters after using distill water dialysis 10h, abandons filtrate again, regathers filter residue, just prepares chitosan-arginine the moon from resin.
(3) Quimotrase is fixing
After (2) step finished, earlier Quimotrase being added the pH value was in 5.9 phosphate buffered saline buffers, stir, and with regard to the volumetric molar concentration of preparing Quimotrase the Quimotrase phosphate solution of 0.06mol/L.The chitosan-arginine resin anion that prepared in (2) step the back: the mass ratio of Quimotrase is 1: 20 a ratio in the Quimotrase phosphate solution, chitosan-arginine resin anion is added in the Quimotrase phosphate solution, under 45 ℃ water bath with thermostatic control condition, vibration 30min is suspended in the Quimotrase phosphate solution chitosan-arginine resin anion.Again in glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 8 a ratio, glutaraldehyde is joined in the Quimotrase phosphate solution of suspension chitosan-arginine resin anion, and place vibrator to vibrate, after carrying out schiff's reaction 60min, carry out suction filtration, abandon filtrate, collect the resin filter residue, the resin filter residue is washed 6 times with phosphate buffered saline buffer, remove free Quimotrase, suction filtration is abandoned filtrate more again, regather chitosan-arginine negatively charged ion immobilizing chymotrypsin filter residue and finished product.
Embodiment two
A kind of concrete steps of method of chitosan-arginine resin anion immobilizing Quimotrase are as follows:
(1) preparation crosslinked chitosan resin
With embodiment one, feature is: it is in 1% dilute hydrochloric acid that chitosan is added the hydrochloric acid mass percent, prepares chitosan mass percentage ratio and be 1% chitosan acidic sol.The chitosan acidic sol: the volume ratio of whiteruss is 1: 2, stir 20min, and heat temperature raising to 35 ℃, add again that to account for chitosan paraffin liquor capacity mark be 0.05% tween 80, carry out emulsification 20min, add then that to account for chitosan paraffin liquor capacity mark be 0.05% glutaraldehyde cross-linking agent, add again that to account for chitosan paraffin liquid quality fraction be 0.01% lime carbonate pore-creating agent, carry out drilling reaction 50min.The pH value of regulating chitosan microball suspension with sodium hydroxide solution is 9, and is heated to 55 ℃, stir to solidify 2h, to filter residue earlier with washing with alcohol 3 times, after be lime carbonate in the 1% diluted hydrochloric acid dissolution filter residue with massfraction.
(2) preparation of chitosan-arginine resin anion
With embodiment one, feature is: the arginine massfraction is 0.5% in the arginine solution, and vibration 20min prepares the chitosan mass mark and be 0.5% chitosan-arginine solution.Arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is 1: 0.5, carries out condensation reaction 20min, and filter residue is used washing with alcohol 3 times earlier, back distill water dialysis 8h.
(3) Quimotrase is fixing
With embodiment one, feature is: earlier Quimotrase being added the pH value is in 4.5 phosphate buffered saline buffers, and the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.05mol/L.The chitosan-arginine resin anion that (2) step prepared: the mass ratio of Quimotrase is 1: 15 in the Quimotrase phosphate solution, under 30 ℃ water bath with thermostatic control condition, vibration 20min, glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 6, carry out schiff's reaction 50min, the resin filter residue is washed 4 times with phosphate buffered saline buffer.
Embodiment three
A kind of concrete steps of method of chitosan-arginine resin anion immobilizing Quimotrase are as follows:
(1) preparation crosslinked chitosan resin
With embodiment one, feature is: it is in 5% dilute hydrochloric acid that chitosan is added the hydrochloric acid mass percent, prepares chitosan mass percentage ratio and be 4% chitosan acidic sol.The chitosan acidic sol: the volume ratio of whiteruss is 1: 4, stir 30min, and heat temperature raising to 45 ℃, add again that to account for chitosan paraffin liquor capacity mark be 0.25% tween 80, carry out emulsification 30min, add then that to account for chitosan paraffin liquor capacity mark be 0.3% glutaraldehyde cross-linking agent, add again that to account for chitosan paraffin liquid quality fraction be 0.1% lime carbonate pore-creating agent, carry out drilling reaction 70min.The pH value of regulating chitosan microball suspension with sodium hydroxide solution is 10, and is heated to 75 ℃, stir to solidify 4h, to filter residue earlier with washing with alcohol 6 times, after be lime carbonate in the 5% diluted hydrochloric acid dissolution filter residue with massfraction.
(2) preparation of chitosan-arginine resin anion
With embodiment one, feature is: the arginine massfraction is 2.0% in the arginine solution, and vibration 30min prepares the chitosan mass mark and be 2.5% chitosan-arginine solution.Arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide (DCC) is 1: 2.5, carries out condensation reaction 40min, and filter residue is used washing with alcohol 6 times earlier, back distill water dialysis 12h.
(3) Quimotrase is fixing
With embodiment one, feature is: earlier Quimotrase being added the pH value is in 6.0 phosphate buffered saline buffers, and the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.08mol/L.The chitosan-arginine resin anion that (2) step prepared: the mass ratio of Quimotrase is 1: 25 in the Quimotrase phosphate solution, under 60 ℃ water bath with thermostatic control condition, vibration 40min, glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 14 a ratio, after carrying out schiff's reaction 70min, the resin filter residue is washed 7 times with phosphate buffered saline buffer.
Experimental result
With the product that embodiment 1～3 prepares, the suitableeest catalytic temperature of chitosan-arginine resin anion immobilizing Quimotrase, the suitableeest catalytic pH value, the enzyme rate of recovery alive, transformation period test have been carried out.Test-results is as follows:
As can be known from the above table, the free Quimotrase height of the thermostability of chitosan-arginine resin anion immobilizing Quimotrase, the suitableeest catalytic temperature of immobilizing chymotrypsin is 70 ℃, free Quimotrase improves 10 ℃; The suitableeest catalytic pH value of immobilizing chymotrypsin is 5.92, and free Quimotrase has reduced by 2.08, has enlarged the use range of immobilizing chymotrypsin; The rate of recovery alive of immobilizing chymotrypsin reaches 18 hours up to the transformation period to 89.95%, 75 ℃, and 4 ℃ transformation period reaches 56 days, has improved the utilising efficiency of immobilizing chymotrypsin, reduces and pollutes, and makes full use of resource.
Claims (4)
1. the method for a chitosan-arginine resin anion immobilizing Quimotrase is characterized in that concrete method steps is as follows:
(1) preparation crosslinked chitosan resin
With commercially available chitosan is raw material, earlier chitosan being added the hydrochloric acid mass percent is in 1～5% dilute hydrochloric acid, just prepare chitosan mass percentage ratio and be 1～4% chitosan acidic sol, back in the chitosan acidic sol: the volume ratio of whiteruss is 1: 1～4 ratio, slowly add the chitosan acidic sol in the whiteruss, stir 20～30min, and after heat temperature raising to 35～45 ℃ make chitosan paraffin solution, add again that to account for chitosan paraffin liquor capacity mark be 0.05～0.25% tween 80, carry out emulsification 20～30min, add then that to account for chitosan paraffin liquor capacity mark be 0.05～0.3% glutaraldehyde cross-linking agent, add again that to account for chitosan paraffin liquid quality fraction be 0.01～0.1% lime carbonate pore-creating agent, carry out drilling and react the suspension that 50～70min makes chitosan microball, pH value with sodium hydroxide solution adjusting chitosan microball suspension is 9～10 at last, and be heated to 55～75 ℃, stir and solidify 2～4h, refilter, abandon filtrate, collect filter residue, filter residue is used washing with alcohol 3～6 times earlier, the back is a lime carbonate in 1～5% diluted hydrochloric acid dissolution filter residue with massfraction, is washed with distilled water to neutrality again;
(2) preparation of chitosan-arginine resin anion
(1) step finish after, go on foot the crosslinked chitosan resin of preparing with (1), adding the arginine massfraction is in 0.5～2.0% arginine solution, vibration 20～30min, prepare the chitosan mass mark and be 0.5～2.5% chitosan-arginine solution, then in arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide is 1: 0.5～2.5 ratio, in chitosan-arginine solution, add dicyclohexylcarbodiimide and connect the peptide condensing agent, carry out condensation reaction 20～40min, at last after filtration, abandon filtrate, collect filter residue, filter residue is used washing with alcohol 3～6 times earlier, and the back refilters after using distill water dialysis 8～12h, abandon filtrate again, regather filter residue;
(3) Quimotrase is fixing
(2) step finish after, earlier Quimotrase being added the pH value is in 4.5～6.0 phosphate buffered saline buffers, stir, with regard to the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.05～0.08mol/L, the chitosan-arginine resin anion that prepared in (2) step the back: the mass ratio of Quimotrase is 1: 15～25 ratio in the Quimotrase phosphate solution, chitosan-arginine resin anion is added in the Quimotrase phosphate solution, under 30～60 ℃ water bath with thermostatic control condition, vibration 20～40min, again in glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 6～14 ratio, glutaraldehyde is joined in the Quimotrase phosphate solution of suspension chitosan-arginine resin anion, and place vibrator to vibrate, after carrying out schiff's reaction 50～70min, carry out suction filtration, abandon filtrate, collect the resin filter residue, the resin filter residue is washed 4～7 times with phosphate buffered saline buffer, suction filtration again, abandon filtrate again, regather chitosan-arginine negatively charged ion immobilizing chymotrypsin filter residue.
2. according to the method for the described chitosan-arginine resin anion immobilizing Quimotrase of claim 1, it is characterized in that concrete method steps is as follows:
In (1) step, it is in 2% dilute hydrochloric acid that chitosan is added the hydrochloric acid mass percent, prepare chitosan mass percentage ratio and be 2% chitosan acidic sol, the chitosan acidic sol: the volume ratio of whiteruss is 1: 1, stir 25min, and heat temperature raising to 40 ℃, add again that to account for chitosan paraffin liquor capacity mark be 0.1% tween 80, carry out emulsification 25min, add then that to account for chitosan paraffin liquor capacity mark be 0.15% glutaraldehyde cross-linking agent, add that to account for chitosan paraffin liquid quality fraction be 0.05% lime carbonate pore-creating agent again, carry out drilling reaction 60min, the pH value of regulating chitosan microball suspension with sodium hydroxide solution is 9.5, and be heated to 60 ℃, stir to solidify 3h, earlier with washing with alcohol 5 times, the back is a lime carbonate in the 3% diluted hydrochloric acid dissolution filter residue with massfraction to filter residue;
In (2) step, the arginine massfraction is 1% in the arginine solution, vibration 25min, prepare the chitosan mass mark and be 1.5% chitosan-arginine solution, arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide is 1: 1, carry out condensation reaction 30min, filter residue is used washing with alcohol 4 times earlier, back distill water dialysis 10h;
In (3) step, earlier Quimotrase being added the pH value is in 5.9 phosphate buffered saline buffers, the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.06mol/L, the chitosan-arginine resin anion that (2) step prepared: the mass ratio of Quimotrase is 1: 20 in the Quimotrase phosphate solution, under 45 ℃ water bath with thermostatic control condition, vibration 30min, glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 8, after carrying out schiff's reaction 60min, the resin filter residue is washed 6 times with phosphate buffered saline buffer.
3. according to the method for the described chitosan-arginine resin anion immobilizing Quimotrase of claim 1, it is characterized in that concrete method steps is as follows:
In (1) step, it is in 1% dilute hydrochloric acid that chitosan is added the hydrochloric acid mass percent, prepare chitosan mass percentage ratio and be 1% chitosan acidic sol, the chitosan acidic sol: the volume ratio of whiteruss is 1: 2, stir 20min, and heat temperature raising to 35 ℃, add again that to account for chitosan paraffin liquor capacity mark be 0.05% tween 80, carry out emulsification 20min, add then that to account for chitosan paraffin liquor capacity mark be 0.05% glutaraldehyde cross-linking agent, add that to account for chitosan paraffin liquid quality fraction be 0.01% lime carbonate pore-creating agent again, carry out drilling reaction 50min, the pH value of regulating chitosan microball suspension with sodium hydroxide solution is 9, and be heated to 55 ℃, stir to solidify 2h, earlier with washing with alcohol 3 times, the back is a lime carbonate in the 1% diluted hydrochloric acid dissolution filter residue with massfraction to filter residue;
In (2) step, the arginine massfraction is 0.5% in the arginine solution, vibration 20min, prepare the chitosan mass mark and be 0.5% chitosan-arginine solution, arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide is 1: 0.5, carry out condensation reaction 20min, filter residue is used washing with alcohol 3 times earlier, back distill water dialysis 8h;
In (3) step, earlier Quimotrase being added the pH value is in 4.5 phosphate buffered saline buffers, the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.05mol/L, the chitosan-arginine resin anion that (2) step prepared: the mass ratio of Quimotrase is 1: 15 in the Quimotrase phosphate solution, under 30 ℃ water bath with thermostatic control condition, vibration 20min, glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 6, after carrying out schiff's reaction 50min, the resin filter residue is washed 4 times with phosphate buffered saline buffer.
4. according to the method for the described chitosan-arginine resin anion immobilizing Quimotrase of claim 1, it is characterized in that concrete method steps is as follows:
In (1) step, it is in 5% dilute hydrochloric acid that chitosan is added the hydrochloric acid mass percent, prepare chitosan mass percentage ratio and be 4% chitosan acidic sol, the chitosan acidic sol: the volume ratio of whiteruss is 1: 4, stir 30min, and heat temperature raising to 45 ℃, add again that to account for chitosan paraffin liquor capacity mark be 0.25% tween 80, carry out emulsification 30min, add then that to account for chitosan paraffin liquor capacity mark be 0.3% glutaraldehyde cross-linking agent, add that to account for chitosan paraffin liquid quality fraction be 0.1% lime carbonate pore-creating agent again, carry out drilling reaction 70min, the pH value of regulating chitosan microball suspension with sodium hydroxide solution is 10, and be heated to 75 ℃, stir to solidify 4h, earlier with washing with alcohol 6 times, the back is a lime carbonate in the 5% diluted hydrochloric acid dissolution filter residue with massfraction to filter residue;
In (2) step, the arginine massfraction is 2.0% in the arginine solution, vibration 30min, prepare the chitosan mass mark and be 2.5% chitosan-arginine solution, arginine in the chitosan-arginine solution: the mol ratio of dicyclohexylcarbodiimide is 1: 2.5, carry out condensation reaction 40min, filter residue is used washing with alcohol 6 times earlier, back distill water dialysis 12h;
In (3) step, earlier Quimotrase being added the pH value is in 6.0 phosphate buffered saline buffers, and the volumetric molar concentration of preparing Quimotrase is the Quimotrase phosphate solution of 0.08mol/L; The chitosan-arginine resin anion that (2) step prepared: the mass ratio of Quimotrase is 1: 25 in the Quimotrase phosphate solution, under 60 ℃ water bath with thermostatic control condition, and vibration 40min; Glutaraldehyde: the mass ratio of chitosan-arginine resin anion is 1: 14, carry out schiff's reaction 70min after, to the resin filter residue with phosphate buffered saline buffer washing 7 times.
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| AU2007257436B2 (en) | 2006-06-02 | 2013-05-02 | Synedgen, Inc. | Chitosan-derivative compounds and methods of controlling microbial populations |
| CN101575384B (en) * | 2009-06-08 | 2011-06-15 | 北京大学 | Nano chitosan derivative and preparation method and application thereof |
| CN102174504A (en) * | 2011-01-14 | 2011-09-07 | 华南理工大学 | Granular carrier immobilized lipase for treating papermaking white water and preparation method thereof |
| CN102199592B (en) * | 2011-04-02 | 2013-04-03 | 重庆大学 | Method for preparing mixed immobilized glucose oxidase/catalase microspheres |
| CN102911928B (en) * | 2012-07-06 | 2014-10-08 | 西南药业股份有限公司 | Immobilized method of creatine kinase |
| CN103451255B (en) * | 2013-03-28 | 2016-03-16 | 南京工业大学 | The production method of inosinic acid |
| CN106477557B (en) * | 2016-11-04 | 2018-12-04 | 江南大学 | A kind of carbon nano tube compound material of aromatic aldehyde/chitosan non-covalent modification |
| CN107522781A (en) * | 2017-09-01 | 2017-12-29 | 浦江县欧立生物技术有限公司 | The method that collagen peptide is extracted from grass carp scales |
| CN109999767A (en) * | 2019-04-17 | 2019-07-12 | 江苏师范大学 | A kind of method of glutaraldehyde modification of chitosan resin adsorption separation purple sweet potato anthocyanin |
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